CN113499332A - Use of MYC agonists for the preparation of a medicament for promoting cell proliferation - Google Patents
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Abstract
The invention relates to the field of biomedicine, in particular to application of a MYC agonist in preparation of a medicine for promoting cell proliferation. The invention provides application of a MYC agonist in preparation of a medicine for promoting cell proliferation, and also provides a cell proliferation regulation method, which comprises the following steps: culturing the cells in the presence of a MYC agonist. The invention provides a compound 2-hydroxy-3-methylbutyric acid (2-hydroxy-3-methylbutyric acid) for promoting cell proliferation, and provides application of the compound in promoting cell proliferation, and related experiments can find that the 2-hydroxy-3-methylbutyric acid has the effect of promoting cell proliferation, so that the compound can be applied to medicines for promoting cell proliferation or regulation and control of cell proliferation, and has an industrial prospect.
Description
Technical Field
The invention relates to the field of biomedicine, in particular to application of a MYC agonist in preparation of a medicine for promoting cell proliferation.
Background
The process of human wound healing is mainly divided into the following steps: inflammatory response, angiogenesis, granulation tissue growth, proliferation and migration of epithelial cells, stem cells and wound healing. Among them, the proliferation of epithelial cells is an important link in the proliferative phase of wound healing, and is closely related to the speed and quality of wound healing.
At present, there are two main mechanisms for promoting cell proliferation: keratinocyte Growth Factor (KGF), an epithelial cell growth factor with strong action and high specificity. Fibroblasts in the base part of the wound can synthesize and release KGF, and induce the cell proliferation around the wound; insulin-like growth factor I (IGF-I), a growth factor protein similar to insulin in humans, promotes carbohydrate metabolism and transport in adipose tissue, fat, glycogen, DNA, RNA, and protein synthesis, ultimately leading to cell proliferation. These cell growth factors are secreted or activated by the injured person, and therefore, the cell proliferation effect produced by the factors is determined by the physical condition of the injured person, such as physical weakness, diabetes, etc., which prolongs the healing period and causes unnecessary time and economic consumption for the injured person. Therefore, the search for new cell proliferation methods is an urgent need for the injured person.
Disclosure of Invention
In view of the above-mentioned drawbacks of the prior art, it is an object of the present invention to provide the use of a MYC agonist for the preparation of a medicament for promoting cell proliferation, for solving the problems of the prior art.
To achieve the above and other related objects, a first aspect of the present invention provides the use of a MYC agonist in the manufacture of a medicament for promoting cell proliferation.
In some embodiments of the invention, the MYC agonist is capable of promoting expression and/or function of a MYC gene.
In some embodiments of the invention, the MYC agonist is a single active ingredient.
In some embodiments of the invention, the MYC agonist is selected from 2-hydroxy-3-methylbutyric acid.
In some embodiments of the invention, the agent that promotes cell proliferation is an agent that promotes cell proliferation by mediating expression of a MYC gene;
and/or, the cells are selected from colon cells, preferably from colon adenocarcinoma cells, more preferably from Caco-2 cells.
In a second aspect, the present invention provides a method for regulating cell proliferation, comprising: culturing the cells in the presence of a MYC agonist.
In some embodiments of the invention, the MYC agonist is capable of promoting expression and/or function of a MYC gene.
In some embodiments of the invention, the MYC agonist is a single active ingredient.
In some embodiments of the invention, the MYC agonist is selected from 2-hydroxy-3-methylbutyric acid.
In some embodiments of the invention, the agent that promotes cell proliferation is an agent that promotes cell proliferation by mediating expression of a MYC gene;
and/or, the cells are selected from colon cells, preferably from colon adenocarcinoma cells, more preferably from Caco-2 cells.
Drawings
FIG. 1 is a schematic diagram showing the chemical structure of 2-hydroxy-3-methylbutyric acid.
FIG. 2 is a diagram showing the results of RNA sequencing in example 1 of the present invention.
FIG. 3 is a diagram showing the results of the fluorescent quantitative PCR in example 1 of the present invention.
FIG. 4 is a schematic diagram showing the results of flow cytometry in example 1 of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments, and other advantages and effects of the present invention will be apparent to those skilled in the art from the disclosure of the present specification.
The inventors of the present invention have surprisingly found, through a large number of practical studies, that 2-hydroxy-3-methylbutyric acid (HMBA) can effectively promote cell proliferation, and thus can be applied to a drug for promoting cell proliferation or cell proliferation regulation, and have completed the present invention.
In a first aspect, the invention provides the use of a MYC agonist in the manufacture of a medicament for promoting cell proliferation. The MYC agonist can effectively improve the expression level of MYC genes in cells, effectively increase the proliferation quantity of Caco-2 cells and reduce the apoptosis rate of the cells, so that the MYC agonist can be applied to medicines for promoting cell proliferation.
In the above uses, the MYC agonist is capable of promoting expression and/or function of a MYC gene. For example, a MYC agonist may generally promote expression and/or function of MYC. As another example, an agonistic function of a MYC agonist can be an increase in expression level at the MYC gene nucleic acid molecule level (e.g., mRNA level, etc.) and/or protein molecule level. Suitable substances capable of acting as MYC agonists may be, for example, 2-hydroxy-3-methylbutyric acid and the like. As mentioned above, a possible molecular mechanism for a drug that promotes cell proliferation may be a drug that promotes cell proliferation by mediating expression of MYC genes.
In the above uses, MYC agonists may be used as the sole active ingredient, or may be used in combination with other active ingredients, together for promoting cell proliferation.
In the above-mentioned applications, the cells to be used may be colon cells, for example, colon adenocarcinoma cells, more specifically Caco-2 cells.
In a second aspect, the present invention provides a method for regulating cell proliferation, comprising: culturing the cells in the presence of a MYC agonist. The MYC agonist can effectively improve the expression quantity of MYC genes in cells, and the Caco-2 cells can effectively improve the proliferation quantity and reduce the apoptosis rate of the cells when being cultured in the presence of the MYC agonist, so that the MYC agonist can be applied to regulation and control of cell proliferation.
In the above method of regulating cell proliferation, the MYC agonist can promote expression and/or function of a MYC gene. For example, a MYC agonist may generally promote expression and/or function of a MYC gene. As another example, an agonistic function of a MYC agonist can be an increase in expression level at the MYC gene nucleic acid molecule level (e.g., mRNA level) and/or protein molecule level. Suitable substances capable of acting as MYC agonists may be, for example, 2-hydroxy-3-methylbutyric acid and the like. As mentioned above, a possible molecular mechanism of the drug that promotes cell proliferation may be a drug that promotes cell proliferation by mediating expression of MYC genes.
In the above cell proliferation regulating method, the MYC agonist may be used as a single active ingredient, or may be used in combination with other active ingredients for promoting cell proliferation.
In the above-mentioned method for controlling cell proliferation, the cells to be used may be colon cells, for example, colon adenocarcinoma cells, more specifically Caco-2 cells.
Among the above-mentioned cell proliferation control methods, suitable cell culture methods should be known to those skilled in the art, and the culture conditions generally correspond to the cell type. For example, it may be at 5% CO2Culturing the cells in the presence of a MYC agonist at 37 ℃.
In a third aspect the invention provides a composition comprising a MYC agonist, for use in: use in a medicament for promoting cell proliferation; and/or, cell proliferation regulation. The MYC agonist described above may be various MYC agonists as described above.
In a fourth aspect, the invention provides a method of treatment comprising: administering to the individual a therapeutically effective amount of a MYC agonist, or a composition as provided by the third aspect of the invention. The treatment provided by the present invention can be used to promote cell proliferation.
In the present invention, "subject" generally includes humans, non-human primates, such as mammals, dogs, cats, horses, sheep, pigs, cows, etc., which would benefit from treatment with the formulation, kit or combined formulation.
In the present invention, a "therapeutically effective amount" generally refers to an amount which, after an appropriate period of administration, achieves the desired effect as set forth above.
The invention provides a compound 2-hydroxy-3-methylbutyric acid (2-hydroxy-3-methylbutyric acid) for promoting cell proliferation, and provides application of the compound in promoting cell proliferation, and related experiments can find that the 2-hydroxy-3-methylbutyric acid has the effect of promoting cell proliferation, so that the compound can be applied to medicines for promoting cell proliferation or regulation and control of cell proliferation, and has an industrial prospect.
The present application is further illustrated by the following examples, which are not intended to limit the scope of the present application.
Example 1
Recovery of Caco-2 cells: frozen Caco-2 cells were taken out of a liquid nitrogen tank, immediately thawed in a water bath at 37 ℃, centrifuged at 1000rpm for 1 minute, the supernatant was removed, the cells were resuspended in MEM culture medium (cell culture medium, Hyclone, USA) containing 20% fetal bovine serum, transferred to a cell culture flask (Corning, USA), 3mL of the cell culture medium was added, and the cells were cultured (5% CO) in a cell culture chamber (HERAcell 240i, thermoelectricity, USA)2And at 37 ℃) for 2 days, taking out, sucking the culture solution, adding fresh cell culture solution, continuing culturing for 3-5 days, performing microscopic examination, and performing subsequent plate paving when the coverage rate of Caco-2 cells on the bottom surface of the culture bottle reaches more than 80%.
Plating of Caco-2 cells: removing the culture medium from the cell culture flask, adding 2mL PBS (Corning, USA), shaking gently, removing residual culture medium, adding 2mL pancreatin (HyClone, USA), covering the bottom surface uniformly, digesting at 37 deg.C for 3 min, taking out, gently blowing with pipette, adding 2mL cell culture medium (stopping digestion), blowing uniformly, collecting culture medium, centrifuging at 1000rpm for 1 min, removing supernatant, adding 1mL cell culture medium, resuspending the cells, measuring the cell number with cell counter (DMEM, USA), diluting the resuspension solution with cell culture medium to 5 × 105Individual cells/mL. Sucking 200 μ L of the above dilution into a 96-well plate, and culturing in a cell culture box for 7 daysAnd (5) changing the culture solution for 1-2 times for later use.
MYC gene up-regulation experiment: selecting a 96-well plate which is qualified in cell culture, taking out culture solution in 3 wells, adding cell culture solution containing 50mM 2-hydroxy-3-methylbutyrate (figure 1, sigma company, USA), selecting 3 wells for the same operation, adding cell culture solution containing PBS with the same volume (blank control), placing in a cell incubator, continuously incubating for 12h, and performing RNA sequencing and fluorescent quantitative PCR detection. RNA sequencing results are shown in figure 2, MYC genes are up-regulated after the Caco-2 cells and 2-hydroxy-3-methylbutyric acid are incubated together, and the up-regulation degree is further quantified through fluorescent quantitative PCR (polymerase chain reaction), and results are shown in figure 3, the expression quantity of the MYC genes in the Caco-2 cells treated by the 2-hydroxy-3-methylbutyric acid is 7 times that of a blank control group, and the results show that the 2-hydroxy-3-methylbutyric acid has a certain stimulation effect on the expression of the MYC genes.
Cell proliferation assay: selecting a 96-well plate which is qualified in cell culture, taking out culture solution in 3 wells, adding cell culture solution containing 50mM 2-hydroxy-3-methylbutyrate (sigma company, USA), selecting 3 wells for the same operation, adding cell culture solution containing the same volume of PBS (blank control), placing the cells in a cell culture box for further incubation for 24h, digesting the cells with pancreatin, and then carrying out flow cytometry detection. The results are shown in FIG. 4, and show that the apoptosis rate of untreated Caco-2 cells is about 13%, while the apoptosis rate of Caco-2 cells treated by 2-hydroxy-3-methylbutyric acid is about 10%, which is significantly lower than the former, and thus, 2-hydroxy-3-methylbutyric acid increases the proliferation amount of Caco-2 cells, thereby reducing the apoptosis rate thereof.
In conclusion, the present invention effectively overcomes various disadvantages of the prior art and has high industrial utilization value.
The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.
Claims (10)
- Use of a MYC agonist in the manufacture of a medicament for promoting cell proliferation.
- 2. The use of claim 1, wherein the MYC agonist is capable of promoting expression and/or function of a MYC gene.
- 3. The use of claim 1, wherein the MYC agonist is a single active ingredient.
- 4. The use of claim 1, wherein the MYC agonist is selected from 2-hydroxy-3-methylbutyric acid.
- 5. The use of claim 1, wherein the agent that promotes cell proliferation is an agent that promotes cell proliferation by mediating expression of a MYC gene;and/or, the cells are selected from colon cells, preferably from colon adenocarcinoma cells, more preferably from Caco-2 cells.
- 6. A method of regulating cell proliferation, comprising: culturing the cells in the presence of a MYC agonist.
- 7. The method for modulating cell proliferation according to claim 1, wherein the MYC agonist is capable of promoting expression and/or function of a MYC gene.
- 8. The method of modulating cell proliferation according to claim 1, wherein the MYC agonist is a single active ingredient.
- 9. The method for modulating cell proliferation according to claim 1, wherein the MYC agonist is selected from 2-hydroxy-3-methylbutyric acid.
- 10. The method for regulating cell proliferation according to claim 1, wherein the agent that promotes cell proliferation is an agent that promotes cell proliferation by mediating expression of a MYC gene;and/or, the cells are selected from colon cells, preferably from colon adenocarcinoma cells, more preferably from Caco-2 cells.
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